1. Field of the Invention
The present invention relates to novel processes for the production of eicosapentaenoic acid and lipid containing eicosapentaenoic acid, by fermentation and using microorganisms belonging to the genus Pseudomonas, Alteromonas or Shewanella.
2. Description of the Related Art
Poly-unsaturated fatty acids represented by eicosapentaenoic acid are important as a component of bio-membranes in an organism. The known pharmacological actions of eicosapentaenoic acid include blood platelet coagulation-inhibition (thrombolytic action), lowering of blood level of neutral fat, lowering of blood levels of VLDL-cholesterol and LDL-cholesterol and increase of blood level of HDL-cholesterol (anti-atherosclerotic action), lowering of blood viscosity, anti-hypertension, anti-inflammation, and anti-tumor actions. Moreover, eicosapentaenoic acid acts as a substrate for the formation of the group of prostaglandins, and thus are essential for higher mammals including humans. In particular, eicosapentaenoic acid is, important as a substrate for the type 3 prostaglandin, inhibits a blood platelet coagulation action, and is promising as a therapeutic and propylactic agent for thrombosis. Moreover, among poly-unsaturated fatty acids responsible for lowering the plasma cholesterol level, eicosapentaenoic acid in particular exhibits a high level of activity in lowering the plasma cholesterol level. Therefore, eicosapentaenoic acid is extremely effective in comparison with other fatty acids, such as linoleic acid, naturally present in plant oil. Eicosapentaenoic acid is also known to be an essential nutrient for fish.
An epidemiological study disclosed by Dyerberg, Am. J. Clin. Nutr. 28, 958, 1975, Denmark suggests the probability of using eicosapentaenoic acid as a pharmaceutical agent and diet food on the basis of the anti-thrombotic action and lipid-lowering action thereof. However, as evident from the structure thereof, a chemical synthesis of eicosapentaenoic acid is very difficult, and therefore, the industrial production of eicosapentaenoic acid is difficult. Under these circumstances, an ingestion of sardine, mackerel, and saury containing a relatively large amount of eicosapentaenoic acid is recommended.
At present, the commercial eicosapentaenoic acid-containing products provided for diet foods are obtained from fish oil prepared by cooking fish, and contain eicosapentaenoic acid in an amount of 10 to 30% by weight. Fish oil prepared by the cooking process comprises a mixture of glycerides which contain various kinds of fatty acids, and therefore, the isolation of eicosapentaenoic acid from the fish oil is difficult. Moreover, since eicosapentaenoic acid is a straight-chain fatty acid with 20 carbon atoms containing five double bonds, all of which are in a cis configuration, it is unstable and susceptible to oxidation. Therefore, the isolation of eicosapentaenoic acid from fish oil must be carried out in such a manner that oxygen, light, and heat are excluded during the process. Moreover, it is difficult to eliminate organic solvents used for the isolation of eicosapentaenoic acid from the product under the usual reduced pressure. Therefore, a complete elimination of the organic solvents is a problem from the technical and economical points of view.
The eicosapentaenoic acid used for pharmaceutical purposes is produced by hydrolysing fish oil extracted by any method, with enzymes, or under an alkaline conditions, to liberate free fatty acids, and optionally, converting the fatty acids to methyl esters or ethyl esters thereof, subjecting the esters to fractional crystallization under a low temperature, urea-addition, distillation under a reduced pressure, reverse phase chromatography or the like, to prepare a product containing at least 90% eicosapentaenoic acid. However, the eicosapentaenoic acid concentrates thus prepared contain organic residual solvents used in the extraction process and have been deteriorated by intermolecular polymerization, isomerization, oxidation or the like. Moreover, eicosapentaenoic acid products produced from fish oil might contain docosenoic acids, which are suspected to be a causal substance of cardio disease, and therefore it is a problem to use them in diet food and pharmaceuticals. Finally, eicosapentaenoic acid products produced from fish oil may emit an unpleasant "fishy" odor.
Recently, to eliminate the above-mentioned disadvantages originating from the use of fish, processes for the production of eicosapentaenoic acid using microorganisms such as chlorella, phaeodactylum, euglena, or algae have been disclosed. For example, J. L. Gellerman and H. Schlenk, BBA, 573, 23, 1979, and Yamada et al, at the Conference of the Fermentation Technology Association of Japan, 1986, reported fungi which produce eicosapentaenoic acid. Such microbial processes for the production of eicosapentaenoic acid are advantageous in that isolation and purification of the target fatty acid from a microbially produced fatty acid mixture is relatively easy, and a preferential production of the target fatty acid in relation to other fatty acids is relatively simple if the fermentation process can be controlled. However, the above mentioned processes require a incubation time of as long as seven days to one month.
Moreover, Summary of the Conference of the Association of Agricultural Chemistry of Japan published on Mar. 10, 1987 discloses production of arachidonic acid and eicosapentaenoic acid by Mortierella.